This project was initiated to study the sequence of events during chemically induced neoplasia using the rodent hepatoma model in combination with quantitative two-dimensional gel electrophoresis (2D-PAGE). Hyperplastic nodules (HN) were generated in male F-344 rats using the resistant hepatocyte model. Six months after initiation animals bearing HN and untreated control rats were treated with the following compounds known to modulate liver enzymes and proteins: lead nitrate (LN), cobaltheme (CoH), 3-methylcholanthrene (3MC), and phenobarbital (PB). In control animals LN, CoH, 3MC, and PB treatment all resulted in a two- to tenfold increase in the expression of the Yc subunit of glutathione-S-transferase (GST). PB also increased the Yb and Ya subunits fivefold each. LN also increased the expression of a polypeptide tentatively identified as the Yp subunit of the placetal form of GST-P. Neither CoH, 3MC, nor PB had any effect on the expression of this polypeptide. Polypeptide 8, composed of 5 isoelectric point variants (6.00-6.60/66,000) was increased two-to threefold in HN from untreated animals and was similarly increased in normal liver following treatment with each of the four modulators. The order of potency was: LN greater than CoH greater than PB = 3MC. Polypeptides 6 (6.60/21,000) and 7 (6.40/16,000) which were expressed at relatively high levels in normal liver (0.5-0.6% of the total integrated density on each gel) were reduced three- to fivefold in HN. Following treatment of normal liver with either LN, 3MC, or CoH, polypeptides 6 and 7 were reduced to 0.1-0.2% and 0.2-0.3%, respectively. Polypeptide 3 (5.90/38,000) which is markedly reduced in HN is similarly decreased in normal liver by LN, PB, and CoH. 2D-PAGE of microsomal polypeptides failed to reveal any common polypeptide changes between HN and modulator-treated normal liver, although numerous qualitative and quantitative differences specific to each modulator were observed.